1. Mechanical Tension Drives Cell Membrane Fusion
Ji Hoon Kim, Yixin Ren, Win Pin Ng, Shuo Li, Sungmin Son, Yee-Seir Kee, Shiliang Zhang, Guofeng Zhang, Daniel A. Fletcher, Douglas N. Robinson, Elizabeth H. Chen 
Developmental Cell 
Volume 32, Issue 5, Pages (March 2015)
DOI: /j.devcel
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2. Developmental Cell 2015 32, 561-573DOI: (10.1016/j.devcel.2015.01.005)
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3. Figure 1
Founder-Cell-Specific Function of Rho1, Rok, and MyoII in Drosophila Myoblast Fusion
(A–H) Stage 15 embryos were labeled with α-muscle MHC antibody. Ventral lateral muscles of three hemisegments are shown in each panel. Anterior is at the left and posterior is at the right. (A) Wild-type (WT). (B and C) Normal myoblast fusion in rho1 (B) and rok (C) mutant. (D) Myoblast fusion defect in rok; rho1 double mutant. (E and F) Expressing a dominant-negative form of Rho1, Rho1N19, in founder cells of WT (E) and rho1 mutant (F) caused myoblast fusion defects. Note the more severe defect in (F) than in (E). (G and H) Expression of a phosphomimetic form of RLC, RLCE21 (G), but not a nonphosphorylatable form, RLCA20,21 (H), rescued the fusion defect in rok; rho1 double mutant. Arrowheads indicate unfused FCMs. Bar, 20 μm.
(I) Quantification of myoblast fusion. The fusion index was determined as the percentage of the number of Ladybird early-positive nuclei in mutant versus WT segmental border muscles (SBMs). Error bars indicate SEM. ∗∗∗p < 10−4.
See also Figure S1 and Table S1.
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4. Figure 2 Localization of Rho1, Rok, and MyoII at the Fusogenic Synapse
Fusogenic synapses (arrowheads) in stage 14 embryos marked by F-actin foci (phalloidin; red) and cell adhesion molecules Duf or Sns (α-Duf or Sns; blue). The attacking FCMs are outlined in the merged panels except for the area of the fusogenic synapse, the plasma membrane within which is impossible to delineate at this resolution.
(A–C′′′) Founder-cell-specific accumulation of Rho1, Rok, and MyoII at the fusogenic synapse. Fluorescently tagged Rho1 (A–A′′′), RokK116A (a kinase-dead form; Simões et al., 2010) (B–B′′′), and Zip (C–C′′′) were specifically expressed in founder cells and visualized by α-GFP staining (green).
(D–F′′′) MyoII activation at the fusogenic synapse. Activated MyoII RLC was visualized by α-phospho-RLC staining (green) (D and F) or by α-Flag staining (green) of founder cell-expressed phosphomimetic RLCE21-Flag (E). Note the enrichment of phospho-RLC and RLCE21 at the fusogenic synapse in wild-type (WT) (D and E) and the markedly reduced accumulation of phospho-RLC in embryo with decreased Rho1 activity (F).
(G–H′′′) RLC phosphorylation is required for its accumulation at the fusogenic synapse. Flag-tagged RLCE21, or nonphosphorylatable RLC, RLCA20, 21, was expressed with the endogenous rlc promoter and visualized by α-Flag staining (G and H). Note the high level accumulation of RLCE21 (G), but not RLCA20,21 (H), at the fusogenic synapse. Bars, 5 μm.
See also Figures S2 and S3.
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5. Figure 3 Rho1 Is Recruited and Activated by Duf upon Sns Binding
(A–A′′′) S2R+ cells coexpressing GFP-Rho1 (green) and Duf-Flag (blue) were mixed with cells expressing Sns-V5 (red). Note the accumulation of Rho1 at the cell-cell contact site (arrowhead). (A′′′′) The relative intensity of Rho1 and Duf along the marked line in (A′′′) was plotted. Bar, 5 μm.
(B–B′′′) S2R+ cells coexpressing GFP-Rho1 (green) and Sns-V5 (red) were mixed with cells expressing Duf-Flag (blue). Note the lack of Rho1 enrichment at the cell-cell contact site (arrowhead). Bar, 5 μm.
(B′′′′) Intensity plot along the marked line in (B′′′).
(C and C′) Increased Rho1 activity in cells coexpressing Rho1 and Duf upon Duf-Sns interaction. (C) Rho1 protein was pulled down by the RBD of Rhotekin. Note the enhanced level of Rho1 pull down when cells coexpressing Duf and Rho1 were mixed with cells expressing Sns. (C′) Quantification of Rho1 pull-down levels from three independent experiments. Error bars indicate SEM.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4
MyoII and Rok Enrichment at the Fusogenic Synapse Is Independent of Duf-Mediated Rho1 Signaling
(A–D′′′) Fusogenic synapses (arrowheads) in stage 14 embryos marked by F-actin foci (phalloidin; red) and DufΔintra (α-Flag; blue). (A–A′′′) Rho1 recruitment to the fusogenic synapse is dependent on the intracellular domain of Duf. GFP-Rho1 was expressed with DufΔintra-Flag in all muscle cells in duf,rst double mutant. Note the lack of Rho1 enrichment at the fusogenic synapse. (B–D′′′) Accumulation of activated MyoII and Rok at the fusogenic synapse in DufΔintra-expressing duf,rst double-mutant embryos. Note the enrichment of GFP-Zip (B), activated RLC (α-phospho-RLC) (C), and Venus-RokK116A (D) at the fusogenic synapse.
(E) The relative intensity of Zip, Rok, and Rho1 enrichment at fusogenic synapses in wild-type and DufΔintra-expressing duf,rst double-mutant embryos. The intensity of fluorescent signal at the fusogenic synapse was compared with that in the adjacent cortical region. Note that in DufΔintra-expressing duf,rst double-mutant embryos, >70% of fusogenic synapses showed significant (>1.5-fold) Zip and Rok enrichment, whereas <20% showed Rho1 enrichment (n > 40 for each protein).
(F–H′′) MyoII and Rok, but not Rho1, accumulate at the fusogenic synapse in the receiving S2R+ cells. Attacking cells expressing Sns and Eff-1 generated F-actin-enriched foci (F′, G′, and H′). The receiving cells expressed Eff-1 and RFP-Zip (F), Venus-RokK116A (G) or GFP-Rho1 (H).
Bars, 5 μm.
See also Figure S4.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 5
MyoII Functions as a Mechanosensor for Cortical Stress Independently of Rho and Rok
(A–G) MyoII accumulation in response to mechanical stress revealed by the MPA assay. (A–F) Representative differential interference contrast (DIC) (left) and fluorescent (right) images of S2 cells aspirated using micropipettes. Fluorescent proteins expressed are indicated above the panels. Note the lack of accumulation of mCherry (A), RokK116A (C), Rho1 (D), and ZipΔmotor (F), but the accumulation of Zip (arrow) in normal (B) and Ca2+-free medium (E). (G) Quantification of protein accumulation at the tip of aspirated cells. Background-subtracted protein pixel intensities at the tip of the cell body within the pipette (lp) and at the opposite pole of the cell body (lo) were measured, and the ratio (lp/lo) was calculated and used for statistical analysis. ∗∗∗p < 10−4. Error bars indicate SEM.
(H–J) MyoII accumulation in response to mechanical stress revealed by AFM. (H) Schematic drawing of the AFM experiments. Cells coexpressing RFP-Zip and GFP-Rho1 (I) or GFP-Zip and RFP- RokK116A (I′) were imaged live over an average time frame of ∼8 min. Stills of the movies are shown. (I) The nudging cantilever induced a rapid accumulation of Zip, but not Rho1, at the sites of deformation. (I′) Zip accumulation in response to the cantilever-imposed force preceded that of Rok. (J) The delay time of Zip mechanosensory response. Note that cells responded rapidly (< 100 s) to the mechanical force imposed by the cantilever.
(K–L′′) The mechanosensory accumulation of MyoII is dependent on its motor domain and the C-terminal BTF assembly domain. RFP- ZipΔmotor or RFP-ZipΔC was expressed in the receiving S2R+ cells treated with Zip dsRNA. Note the absence of any mechanosensory accumulation of either Zip mutant (K and L).
(M–N′′) A positive feedback loop between Rok and MyoII. RFP-Zip or Venus-RokK116A was expressed in the receiving S2R+ cells treated with Rok or Zip dsRNA. The invasive F-actin foci were marked with phalloidin staining (green in M′ and M′′; red in N′ and N′′). Note the absence of Zip or Rok accumulation in Rok (M–M′′) or Zip (N–N′′) knockdown cells.
Bars, 5 μm.
See also Movies S1 and S2.
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 6
MyoII Activity Increases Cortical Resistance Required for Fusion Pore Formation
(A–H) Deeper PLS invasion in embryos with reduced MyoII activity. (A–E′) Confocal images of F-actin foci labeled by phalloidin staining in wild-type (WT) (A and A′), rok; rho1 (B and B′), founder cell::Rho1N19; rho1 (C and C′), dufrP (D and D′), and dufrP; zip (E and E′) embryos. Muscle cell adhesion sites labeled with α-Duf (blue) and FCMs are outlined by dashed lines. Note the roundish morphology of the F-actin focus in WT (A) and dufrP (D) but the wider (B) and deeper (B, C, and E) protrusions in mutant embryos. Arrowheads indicate the tips of invasive protrusions. (F–H) Electron micrographs of the invasive PLSs in WT (F) and founder cell::Rho1N19; rho1 (G and H) embryos. FCMs invading founder cells are pseudocolored in pink. The F-actin-enriched areas are demarcated by dashed lines, based on the relatively low amount of ribosomes and/or intracellular organelles in these areas compared with the rest of the cell body. Note the wider (G) and deeper (G and H) protrusions, as well as the increased amount of ribosomes (G and H) and intracellular organelles (H) within the protrusions.
(I–I′′′) Fusion pores fail to form between muscle cells with reduced MyoII activity. Cytoplasmic GFP was coexpressed with Rho1N19 in founder cells of rho1 mutant embryos stained with α-GFP (green), phalloidin (red), and α-muscle MHC (blue). Note that GFP in miniature myotubes (green in I and I′′′) did not diffuse into the attached FCMs (arrows in I′′ and I′′′), which invaded into the myotube with deep protrusions (arrowheads in I′ and I′′′).
(J) The intensity of GFP signals in myotubes versus the attached, mononucleate FCMs was quantified (n = 22 myotube-FCM pairs). Error bar indicates SEM.
Bars: (A–E and I) 5 μm; (F–H) 500 nm.
Developmental Cell  , DOI: ( /j.devcel )
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9. Figure 7
Artificially Increasing Cortical Tension in Receiving Cells with Decreased MyoII Activity Rescues the Fusion Defect and Models Describing the Mechanosensitive Accumulation of MyoII and the Function of Chemical Signaling in Cell-Cell Fusion
(A and B) AFM analysis of cortical stiffness. (A) Schematic drawing of the AFM experiments. (B) Measurement of cortical stiffness of S2 cells expressing Zip dsRNA and/or Fimbrin (Fim). KD, knockdown; OE, overexpression. ∗p < 0.05 and ∗∗p < Error bars indicate SEM.
(C–G) Fim overexpression rescued the fusion defect caused by Zip KD in the receiving cells. (C–F) Schematic representations and confocal images of cell-cell fusion in S2R+ cells. Attacking cells expressing Sns, Eff-1, and UAS-mCherry were mixed with receiving cells expressing Eff-1, ubiquitin (Ub)-GAL4, and Zip dsRNA (D and F) or Venus-Fim (E and F). Cells were stained with DAPI (nuclei; blue) and phalloidin (F-actin; green) (C and D). (G) Statistical analysis of cell fusion. The fusion index was calculated as percentage of the average nuclei number in mCherry-positive syncytia (n > 65) in (D), (E), or (F) versus that in (C). Fusion between attacking and receiving cells was indicated by mCherry expression in the multinucleate syncytia (red). Bars, 5 μm.
(H–K) Fim overexpression in founder cells significantly rescues the fusion defect in embryos with decreased MyoII activity. Stage 15 founder cell::Rho1N19; rho1 embryos were labeled as in Figure 1. Arrowheads indicate unfused FCMs. The fusion index was quantified in (K). Bar, 20 μm. Error bars indicate SEM. ∗∗∗p < 10−4.
(L) Cortical deformation by PLS invasion induces MyoII accumulation. Prior to PLS invasion, the cortical actin network is under less tension and only a few MyoII BTF are present. During PLS invasion, the protrusive force from the attacking cell deforms the cortical actin network in the receiving cell. Actin network deformation, in turn, applies load to the bound MyoII BTFs and cause MyoII stalling on the strained actin filaments. More BTFs then cooperatively bind to these strained actin filament, ultimately leading to the accumulation of MyoII in response to the mechanical stress.
(M) Rho1 signaling mediated by cell adhesion molecules enhances MyoII activation at the fusogenic synapse. In the absence of Duf-mediated Rho1 accumulation/activation at the fusogenic synapse, MyoII is activated by the basal level of Rok in the cytoplasm and forms a feedback loop with Rok. In the presence of Duf-mediated Rho1 signaling, more freely diffusible MyoII are phosphorylated and activated, providing additional BTFs for binding to strained actin network.
See also Figure S5.
Developmental Cell  , DOI: ( /j.devcel )
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